The virus family Paramyxoviridae contains a number of important human pathogens, including respiratory syncytial virus (RSV), parainfluenza viruses, measles, mumps, and Nipah viruses. This project will involve work on RSV and parainfluenza virus type 3 (PIV-3). Together these viruses are responsible for the majority of cases of pediatric respiratory tract disease and inflict a significant economic and emotional burden. The focus of this project is to understand the mechanism by which the paramyxovirus polymerase accurately initiates genome replication to avoid loss of terminal nucleotides. Paramyxoviruses have a genome consisting of a single strand of negative sense RNA, which is replicated by the virus encoded RNA dependent RNA polymerase. Studies with RSV have shown that the first nucleotide of the replication product is selected independently of the template. This finding, in combination with inspection of paramyxovirus promoter sequences, suggests a model in which the paramyxovirus polymerase becomes preloaded with the first two nucleotides of the replication product prior to its interaction with the template. The aim of this project is to test this hypothesis by addressing three aims. (1) Reverse-genetics studies will be performed to determine if nucleotide 2 of the RSV replication product is selected in a template independent manner, similarly to nucleotide 1. (2) Biochemical experiments will be carried out to determine if the RSV polymerase associates specifically with nucleotides 1 and 2 and generates a dinucleotide primer in the absence of a template. (3) Experiments will be carried out on PIV-3 to determine if initiation mechanisms are conserved across the Paramyxoviridae. The finding that the RSV polymerase can select the initiating nucleoside triphosphate independently of the template is a highly novel result. Understanding the mechanism by which this occurs will provide information regarding a key step in the RSV replication cycle and the work performed in this study will indicate if this mechanism is conserved across the paramyxovirus family. In the long-term, this work could allow design of inhibitors that specifically target the paramyxovirus initiation complex. PUBLIC HEALTH RELEVANCE: The paramyxovirus family of viruses includes a number of important human pathogens. The aim of this project is to understand how the virus initiates replication to produce progeny genomes, a critical step in the virus multiplication cycle. Understanding this step could provide information that could be used to develop antiviral drugs against the viruses.